Auotimmune myocarditis, which can occur after viral infection, underlies many cases of dilated cardiomyopathy. CD8+ cytolytic T lymphocytes mediate much of the damage in myocarditis, but the mechanisms by which naive CD8+ T cell tolerance is broken, and the regulation of autoreactive effector CD8+ T cells are incompletely understood. In this proposal, a newly developed transgenic model of myocarditis will be used to study the regulation of heart antigen-specific CD8+ T cells and to determine how several T cell costimulatory and inhibitory pathways influence disease. Mice that express the model antigen ovalbumin (cMy-mOva) will be used in conjunction with ovalbumin peptide-specific CD8+ T cells from the OT-1 TCR transgenic mouse. Adoptive transfer of activated OT-1 cells, or naive OT-I cells followed by infections with ovalbumin-expressing virus, causes myocarditis in cMy-mOva-ova mice. Disease can be assessed by histology, serum troponin levels, ultrasonography, and mortality. There are three Specific Aims in this proposal. Specific Aim 1. Determine the role of CTLA-4 in regulation of heart-antigen specific CD+ T cells. CTLA-4 is a negative regulator of T cell activation, but little is known, about the role of this molecule in inhibiting autoreactive CD8+ T cells. In this Aim, the pathogenic potential and in vivo fate of CTLA-4+/+ and CTLA-4 OT-I cells in alpha MHC-ova mice will be compared. Specific Aim 2. Determine the role of the PD-1:PD-L 1/2 pathway in CD8+ T cell-mediated myocarditis PD-1, a newly identified member of the CD28 family, inhibits effector functions of T cells when it binds its ligands PD-L1 and PD-L2. The role of this pathway in negatively regulating heart-antigen specific CD8+ T cells in vivo will be explored using PD-L1/L2 -/- cMy-mOva mice as recipients of OT-I cells. Specific Aim 3. Determine the role of ICOS in CD8+ T cell-mediated myocarditis. ICOS, a member of the CD28 family of costimulatory molecules, may have particular importance in sustaining activation of effector T cells. In this Aim, the pathogenic potential, effector functions, and in vivo fate of ICOS -/- and ICOS +/+ OT-I cells will be compared in the cMy-mOva mouse model of myocarditis. This project will provide new information about the regulation of autoreactive CD8+ T cells, and will clarify how CD8+ T cell responses against myocardial antigens are controlled. The focus is on newly characterized T cell regulatory pathways, which are potential targets for immunotherapy of myocarditis and other autoimmune diseases.